Angiogenesis is the process of new blood vessel formation from pre-existing vessels, involving endothelial cell proliferation, migration and assembly into tubule structures (31). It plays important roles in many normal physiological functions such as embryonic development and wound healing (1). In addition, inappropriate angiogenesis is also associated with various pathological conditions including solid tumor growth and metastasis, rheumatoid arthritis and psoriasis (2). Many molecules that inhibit tumor angiogenesis have been shown to inhibit tumor growth including antibodies against angiogenic factors, natural and synthetic compounds that inhibit angiogenesis, and the natural angiogenic inhibitors like the angiostatin and endostatin proteins produced by tumor cells (3-8). Anti-cancer therapy by inhibiting tumor angiogenesis is called anti-angiogenic therapy and has shown great potential as an effective new method for treating cancer, especially solid tumors (9).
Plasminogen is a plasma glycoprotein synthesized mainly in the liver. It is the precursor of the serum protease plasmin that plays important roles in the fibrinolytic system and clot dissolution (10). At the amino terminus, plasminogen contains five homologous repeats that form looped “kringle” structures held together by disulfide bonds. Plasminogen binds to fibrin through lysine binding sites located on the five kringle domains (k1 through k5) (10). Each kringle domain is about 80 amino acids in length and different kringle domains are highly homologous to each other in amino acid sequences. A naturally occurring fragment of plasminogen containing the first four kringle domains (k1-k4) has been isolated from serum and urine of mice bearing a low metastic Lewis lung carcinoma. This plasminogen fragment has been named angiostatin (equivalent to amino acids 98-440 of murine plasminogen) and is a potent angiogenesis inhibitor that can inhibit endothelial cell proliferation as well as tumor growth and metastasis in mice with no obvious toxicity (5, 6, 11). Furthermore, recombinant individual kringle domains or their combinations expressed in E. coli have been found to be able to inhibit endothelial cell proliferation to various degrees with k5 the most potent, followed by k1 and k3 (12, 13). The k5 domain is not present in the naturally existing angiostatin protein but recombinant k5 also functions as an angiogenic inhibitor by inhibiting endothelial cell proliferation, migration as well as inducing cell cycle arrest and apoptosis (13, 32, 33). All these studies indicated that the integrity of the kringle structures and its proper folding are critical in maintaining the kringle domain's as well as angiostatin's functions as angiogenic inhibitors.
Endostatin is a protein first identified from a hemangioendothelioma cell line in 1997 (17). It is a 20 kDa C-terminal fragment of collagen XVIII, a novel collagen that consists of a N-terminal region, a series of collagen-like domains with interruptions and a 35 kDa C-terminal noncollagenous domain (18,19,20). Recombinant endostatin functions as a potent angiogenesis inhibitor in vitro as well as in vivo (17). Systemic administration of endostatin to tumor bearing mice regressed the primary tumor without inducing drug resistance (21). Recently, endostatin was found to be a zinc-binding protein and the zinc-binding is essential for its antiangiogenic activity (22, 23). Human clinical trials of endostatin started in September, 1999.
Vascular Endothelial Growth Factor (VEGF) is a potent endothelial specific mitogen. VEGF functions through two high affinity tyrosine kinase receptors: FLT-1 or Vascular Endothelial Growth Factor Receptor Type 1 (FLT-1/VEGFR1) and FLK-1, also known as KDR or Vascular Endothelial Growth Factor Receptor Type 2 (FLK-1/VEGFR2) (26). Both receptors are specifically expressed in endothelial cells. FLT-1 and KDR/FLK-1 stimulate endothelial cell proliferation and migration by binding to these two tyrosine kinase receptors (24). VEGF is also known as vascular permeability factor due to its ability to induce vascular leakage (24). The ligand binding domains of the two receptors as well as the receptor binding sites of VEGF have been studied by site-directed mutagenesis and X-ray crystallography (25-28). VEGF binds its two receptors through different amino acid contacts (25). The first three immunoglobulin loops of the FLT-1 receptor seem to be the main area responsible for VEGF binding (29, 30). The signal transduction triggered by VEGF through its receptors play critical roles in both physiological angiogenesis as well as pathological angiogenesis such as solid tumor growth and metastasis by stimulating embryonic angiogenesis and tumor angiogenesis. An anti-VEGF monoclonal antibody has been shown to inhibit tumor growth in mice by reducing the vessel density of the tumor (14). Likewise, trans-dominant mutants of both receptors have been shown to inhibit tumor growth in mice (15).
Protein-protein interactions are crucial to many physiological and pharmacological processes. They are specific and exhibit high affinity interactions due to molecular recognition sites found on the surface. It has also been observed that proline residues are sometimes found at the ends of the linear sequences that constitute the site of a protein-protein interaction. It has been shown that the probability of finding proline residues in the flanking segments of the protein-protein interaction sites is 2-3 times that of their random distribution. And the proline residues are not normally present within the interaction sites, but in the flanking segments. They are not directly involved in the interaction between proteins (14, 15).